CLUSTERING AND MOBILITY OF VOLTAGE-DEPENDENT SODIUM-CHANNELS DURING MYELINATION

In myelinated axons, voltage-dependent sodium channels are segregated at high density at nodes of Ranvier (Rosenbluth, 1976; Waxman and Quic k, 1978; Black et al., 1990; Elmer et al., 1990), a distribution that is critical for the saltatory conduction of action potentials (Huxley and Stampfli, 1949). The factors that specifically control the organiz ation and immobilization of sodium channels at nodes are unknown. Rece ntly we have reported that segregation of sodium channels on axons is highly dependent on interactions with active Schwann cells and that co ntinuing axon-glial interactions are necessary to maintain sodium chan nel distribution during differentiation of myelinated nerve (Joe and A ngelides, 1992). The specific recruitment of sodium channels at these early stages of myelination and the conspicuous absence of other axon membrane components suggest that the factors governing sodium channel cluster formation show molecular specificity. However, it is not clear whether these clustered sodium channels originate from a redistributi on of preexisting diffusely distributed sodium channels. To determine how Schwann cells might regulate sodium channel distribution during my elination we have examined the lateral mobility of fluorescently label ed sodium channels at defined stages of myelination by fluorescence ph oto-bleach recovery using tetramethylrhodamine (TmRhd)-labeled Tityus gamma, a sodium channel-specific fluorescent toxin. First, to test whe ther Schwann cells, in addition to modulating sodium channel distribut ion, affect the mobility of sodium channels, we cultured dorsal root g anglion neurons in the presence or absence of Schwann cells and monito red sodium channel mobility on cell bodies, axon hillocks, and axons. Even in the absence of Schwann cells, approximately 80% of the sodium channels were immobile on the time scale of the fluorescence photoblea ch recovery measurement (D(L) less-than-or-equal-to 10(-12) cm2/sec), although the remaining fraction of channels are mobile with diffusion coefficients of 5-13 x 10(-11) cm2/sec. Most importantly, in contrast to the effects of Schwann cells on altering the distribution of sodium channels, we found that Schwann cells did not alter the rate of later al mobility or the mobile fraction of axonal sodium channels. Therefor e, although sodium channel distribution depends on Schwann cell contac t, immobilization of sodium channels is independent of Schwann cell co ntact. These effects appear to be specific for sodium channels because 45% of the succinyl concanavalin-A receptors on the axon are mobile, a fraction that decreases to 25% in the presence of Schwann cells late r in development. To determine how sodium channels might be immobilize d other than by Schwann cell contact, TmRhd-Tityus gamma-labeled dorsa l root neurons were treated with 0.5% Triton X-100. Under these condit ions, 17% and 22% of sodium channels were extracted from cell bodies a nd axons, respectively, suggesting that the immobile sodium channels a re linked to the cytoskeleton. Consideration of several mechanisms sho ws that the results are consistent with the possibility that sodium ch annel clusters arise by the lateral diffusion and trapping or selectiv e exclusion of the small mobile and homogeneously distributed populati on of sodium channels and/or by localized insertion of newly synthesiz ed channels that preferentially appear on the axon at sites of Schwann cell contact. These changes in sodium channel distribution are subseq uently stabilized and maintained by interactions with the subaxolemmal cytoskeleton.